We propose in vitro studies of the kinetics of formation of native three-dimensional structure in proteins, to find physiologically feasible conditions for rapid structure formation, and to obtain information on the mechanism(s) of the self-assembly process. The identity and distribution of disulfide cross-linked intermediates will be determined at various stages of the regeneration of reduced, disorganized proteins. Limited proteolysis followed by column chromatographic separations make it possible to separate disulfide-containing peptides, which will be identified by amino acid composition. The time-pattern of formation of disulfides outlines a folding pathway or a manifold of such pathways. In some cases additional information on the folding pathway may be obtained from parallel kinetic study of the circular dichroic spectra and the fluorescence emission spectra. We will test the ability of fragments of proteins to form native-like structures from unordered structures. Native-like structures will be evaluated by disulfide-bonding pattern, catalytic and/or binding functions, and immuno-chemical cross-reactivity. In cases where fragments do fold to native-like structures, kinetic studies of the folding will be carried out. The enzymic activity of the lysozyme forms as a transient intermediate in hot glutathione regeneration solution. Mechanistic studies of this phenomenon, and testing of its possible generality with other proteins, will be pursued.